The Invasin protein from Yersinia is a outer surface protein that induces phagocytosis of the pathogenic Yersinia by mammalian cells in a beta integrin dependent manner(Wiedemann et al., 2001). Whereas the Listerolysin O cytolysin of Listeria moncytogenes is a well documented cytolysin, which allows for lysis of the endosome vacuole by the pathogen after entry into a mammalian cell (Dramsi & Cossart, 2002). This facilitates endosomal escape of the bacteria. When these two [proteins are coupled together, they can confer non-pathogenic bacteria with suitable features to serve as a drug delivery vector to the cytoplasm.

In constructing our Anaerobic Response System, we chose Invasin and Listeriolysin as part of our drug delivery system, building upon a construct from the Warsaw 2010 team, BBa_K299812. BBa_K299812 is a useful intermediate part containing the Invasin and Listeriolysin with ribosome binding sites in front of the genes. Different promoter sequences could be prepended ahead of the part and modified for our use.

However, when sequencing the part we received from the repository, our team found that the part sequence had major mismatches with the documented sequence. This was particularly serious for the invasin gene, which had only 55% identity between the translated amino acid sequences from the part wiki and our sequencing results. Furthermore, even though BLAST results showed that the invasin gene was indeed present, the strain of origin seemed to be different from the documentation. Lastly, the part does not strictly adhere to BioBricks standard, because the suffix sequence had only the PstI site left. (Further details and updated sequence can be found here.)

As our lab only has the resources to functionally characterise the effect of the expression of invasin through an invasion assay, invasin was our main focus for the construction of parts for our Anaerobic Response System.

Dramsi, S. and Cossart, P. (2002) Listeriolysin O: a genuine cytolysin optimized for an intracellular parasite. J Cell Biol. 2002 Mar 18; 156(6): 943–946.

Wiedemann A1, Linder S, Grassl G, Albert M, Autenrieth I, Aepfelbacher M. (2001) Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages. Cell Microbiol. 2001 Oct;3(10):693-702.